We are examining the relationship between the structural and kinetic properties of three DNA polymerases and their fidelity. Having previously characterized the proofreading-deficient Klenow polymerase in detail, this year we focused on four Klenow mutators. The base substitution mutators reveal three fingers and palm residues that are key determinants of nucleotide selectivity. A frameshift mutator polymerase results from deletion of amino acids in the tip of the polymerase "thumb" domain that contacts the minor groove of the duplex template-primer. Thus, the two major pathways by which errors are initiated (misinsertion and strand slippage) are partly controlled by different polymerase subdomains. This year we completed the first study of mutant forms of beta polymerase based on the X-ray crystal structure of a ternary enzymeDNAdNTP complex. We substituted an arginine residue that contacts the active site template nucleotide with alanine, obtaining the least accurate DNA polymerase ever found. This study strongly suggests that beta pol reads proper Watson-Crick geometry in the active site by probing the position of hydrogen bond donor and acceptor groups in the active site minor groove. Based on the rich structural information available for this polymerase, a variety of additional studies are underway to further probe this and several other concepts for how polymerases accurately copy DNA. It is our belief that structure function studies of DNA polymerases will improve our understanding of how the human genome is stably replicated and maintained, and how DNA adducts affect gemone stability.